Methods for producing isomers of muconic acid and muconate salts

a technology of muconate salt and isomer, which is applied in the field of biological production of muconate to achieve the effects of reducing the number of isomers, improving the solubility of isomers, and improving the purity of isomers

Active Publication Date: 2014-08-19
AMYRIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]In various embodiments of the invention, after the cis,cis-muconate has been produced, it can subsequently be isomerized to cis,trans-muconate or trans,trans-muconate, both of which have differing physical properties and chemical reactivity which can give utility different from or beyond that of cis,cis-muconate. For example, cis,trans-isomer can have greater solubility than cis,cis-muconate in aqueous and / or organic media, allowing advantageous recovery and processing. As a further example, the trans,trans-isomer can have unique utility over the cis,cis-isomer as a reactant in Diels-Alder reactions.

Problems solved by technology

However, as the terms “muconic acid” and “muconate” refer to the protonated or deprotonated forms of the same molecule, the terms are used synonymously where the difference between protonated and deprotonated (e.g., non-ionized and ionized) forms of the molecule is not usefully distinguished.

Method used

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  • Methods for producing isomers of muconic acid and muconate salts
  • Methods for producing isomers of muconic acid and muconate salts
  • Methods for producing isomers of muconic acid and muconate salts

Examples

Experimental program
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example 1

Cloning of the aroZ Gene

[0085]The gene encoding DHS dehydratase, designated aroZ, was isolated from a genomic library of Klebsiella pneumoniae DNA. Genomic DNA was purified from K. pneumoniae strain A170-40 and partially digested with BamH I to produce fragments in the range of 15 kb to 30 kb. The resulting DNA fragments were ligated to cosmid pLAFR3 which had previously been digested with BamH I and subsequently treated with calf intestinal alkaline phosphatase. pLAFR3 is a tetracycline resistant cosmid possessing the RK2 replicon. Ligated DNA was packaged using Packagene Packaging System (Promega), and the resulting phage particles were used to infect E. coli DH5α / pKD136. Plasmid pKD136 is a pBR325-based vector (pMB1 origin of replication) containing genes which encode transketolase (tkt), DAHP synthase (aroF), and DHQ synthase (aroB) as well as an ampicillin resistance gene. Colonies which were resistant to both tetracycline and ampicillin were subsequently plated onto chromogeni...

example 2

Confirmation of the Cloning of the aroZ Gene

[0086]Confirmation that cosmid p5-87 contained the aroZ gene relied on the fact that transformation of an E. coli strain which typically converts D-glucose into DHS could further convert DHS into protocatechuic acid. E. coli AB2834 accumulates DHS in the culture supernatant due to a mutation in the aroE gene, which encodes shikimate dehydrogenase. Conversion of D-glucose to DHS is maximized when AB2834 is transformed with pKD136. AB2834 was co-transformed with pKD136 and p5-87 to produce colonies that were resistant to both ampicillin and tetracycline. One liter of LB medium (4 L Erlenmeyer flask) was inoculated with an overnight culture (5 mL) of AB2834 / pKD136 / p5-87. The culture was grown at 37° C. for 8 h with agitation (250 rpm). The cells were then harvested and resuspended in one liter (4 L Erlenmeyer flask) of minimal M9 medium containing glucose (10 g L), shikimic acid (0.04 g L), ampicillin (0.05 g L), and tetracycline (0.013 g L)....

example 3

Subcloning of the aroZ Gene

[0087]In an effort to minimize the size of the aroZ-encoding insert plasmid p5-87 was digested with BamH I and the resulting fragments were ligated to vector pSU19 which had previously been digested with BamH I and treated with phosphatase. Plasmid pSU19 contains the p15A replicon and the gene which imparts resistance to chloramphenicol. Following transformation of the ligation products into E. coli DH5α / pKD136, the resulting ampicillin and chloramphenicol resistant colonies were screened as described in Example 1 for the ability to turn chromogenic minimal medium agarose plates containing p-toluidine and ferric citrate brown. Using this technique, plasmid pSU1-31 was isolated which consisted of a 3.5 kb BamH I insert contained in pSU19. When AB2834 / pKD136 / pSU1-31 was grown on a 1 L scale under conditions similar to those described in Example 1, 1H NMR analysis of the culture supernatant of indicated that 11 mM protocatechuic acid accumulated extracellular...

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Abstract

A method for producing cis,trans- and trans,trans-isomers of muconate by providing cis,cis -muconate produced from a renewable carbon source through biocatalytic conversion; isomerizing cis,cis-muconate to cis,trans-muconate under reaction conditions in which substantially all of the cis,cis-muconate is isomerized to cis,trans -muconate; separating the cis,trans-muconate; and crystallizing the cis,trans-muconate. The cis,trans-isomer can be further isomerized to the trans,trans-isomer. In one example, the method includes culturing recombinant cells that express 3-dehydroshikimate dehydratase, protocatechuate decarboxylase and catechol 1,2-dioxygenase in a medium comprising the renewable carbon source and under conditions in which the renewable carbon source is converted to 3-dehydroshikimate by enzymes in the common pathway of aromatic amino acid biosynthesis of the cell, and the 3-dehydroshikimate is biocatalytically converted to cis,cis-muconate.

Description

RELATED APPLICATIONS[0001]This application is a National Phase application of International Application No. PCT / US2011 / 020681 filed Jan. 10, 2011, which claims the benefit of and priority to U.S. Provisional Patent Application No. 61 / 335,638, filed Jan. 8, 2010, the disclosures of each of which applications are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The invention relates generally to the biological production of muconate from renewable feedstock. The invention relates more particularly to the production of muconate isomers, as well as precursors and derivatives thereof, from a renewable biomass-derived carbon source.BACKGROUND OF THE INVENTION[0003]Worldwide consumption of dimethyl terephthalate (DMT) is projected to average to 3.97 million metric tons by 2012. DMT is an ester of terephthalic acid and methanol and is used in the production of polyesters, including polyethylene terephthalate and polytrimethylene terephthalate. DMT is also a pri...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07C51/353C12N9/02C12N9/88C12P7/44
CPCC12N9/88C12N9/0069C12P7/44
Inventor BUI, VULAU, MAN KITMACRAE, DOUGSCHWEITZER, DIRK
Owner AMYRIS INC
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